Various types of bacteria assist in transforming milk into other foods. In particular, acid-forming bacteria are used in processes for making yogurt, cheeses, buttermilk, sour cream, and other fermented dairy products. Controlling the growth of the bacteria used in producing such foods is important for ensuring edibility and flavor, as well as for preventing excessive curdling and the occurrence of undesirable secondary fermentations.
The present invention is directed to various agents for controlling the growth of bacteria. The anti-bacterial agents described herein are bacterially produced proteins known as "bacteriocins". Bacteriocins are generally defined by three criteria--(1) they are proteins or a complex of proteins; (2) they are active against bacteria closely related to the producer bacterium; and (3) they kill sensitive bacteria by some means other than lysis.
Bacteriocins were first reported in 1925. Filtrates of a particular strain of E. coli were observed to strongly inhibit the growth of another strain of E. coli. The inhibitory substance was found to be heat-resistant, diffusible through cellophane membranes, and not antigenic. Various other bacteriocins have also been reported in many other gram-positive and gram-negative species.
Bacteriocins are generally divided into two groups: (1) the "true" bacteriocins; and (2) the bacteriophage-like bacteriocins. True bacteriocins lack a complex structure and have an upper limit molecular weight of 400,000 daltons. The true bacteriocins are distinguishable from bacteriophage-like bacteriocins by electron microscopy analysis, during which they appear to be spherical particles ranging from 8 nanometers to 64 nanometers in diameter.
Bacteriophage-like bacteriocins are generally classified into three main groups: (1) tailed particles with full or empty heads that resemble ordinary bacteriophages of Bradley groups A1, B1, B2, or C1; (2) "killer particles" that have small heads and long contractile tails and contain bacterial DNA; and (3) bacteriophage tails that may or may not be contractile. True bacteriophages are viral organisms that destroy bacteria by disintegration and dissolution (lysis).
The nomenclature of bacteriocins is based on the specific name of the host organism. Thus, bacteriocins of E. coli are colicins, Listeria monocytogenes strains produce monocins, and Bacillus cereus strains produce cerecins.
The bacteria genus Propionibacterium, from which the presently described bacteriocins are produced, includes gram-positive nonsporeforming pleomorphic rods. The propionibacteria are nonmotile, have clumps that resemble "Chinese characters" produce large amounts of propionic and acetic acids as fermentation products, are generally catalase-positive, and grow best under anearobic conditions from about 30.degree. C. to 37.degree. C. There are two major groups in the genus Propionibacterium: (1) the acnes group or cutaneous strains; and (2) the classical or dairy strains.
The acnes strains are typically found on human skin. They were originally included in the genus Corynebacterium but were transferred to the genus Propionibacterium because they were anaerobic, produced propionic acid as a major end product of metabolism, contained L-diaminopimellic acid in their peptidoglycan, produced C.sub.15 iso- and anteiso-acids in cell lipids, and lacked mycolic acids and arabinoglycan, which are characteristics of Corynebacterium. The acnes strains include P. acnes, P. avidum, P. granulosum, and P. lymphophilum.
Classical species include Propionibacterium jensenii, P. acidipropionici, E. freudenreichii subspecies freudenreichii and shermanii, and P. thoenii. These classical species are extremely useful organisms in the food industry and are found in dairy fermentations, other natural fermentations such as silage and olives, and soil. During fermentative metabolism, they convert glucose and lactate to propionate, acetate, and carbon dioxide. The inhibitory effects of propionate and acetate are potentiated by the low pH encountered in Swiss cheese and other fermented products. The major use of such classical propionibacteria is in the production of Swiss cheese, where production of carbon dioxide, propionate and acetate by Propionibacterium shermanii (in conjunction with Streptococcus thermophilus and Lactobacillus bulgaricus) contributes the characteristic flavor, texture, and "eyes" of Swiss cheese.
The propionibacteria also produce large amounts of propionic acid which is used for grain preservation, making of cellulose plastics, and in herbicides. In addition, propionic acid and its salts are incorporated into bakery products to prevent mold growth and ropiness. Although the vast majority of propionic acid in the United States is produced by chemical synthesis, research is underway to improve the economics of propionic acid production by fermentation.
Another industrial product of propionibacteria is Microgard.TM. made by Wesman Foods, Inc., of Beaverton, Ore. Microgard.TM. is an FDA-approved and patented product produced by fermenting grade A skim milk with Propionibacterium shermanii followed by pasteurizing. Microgard.TM. is a small heat-stable molecule having a size of about 700 daltons and is antagonistic to most gram-negative bacteria, as well as some yeasts and molds, but not to gram-positive bacteria. It is used as a preservative in about 30% of the cottage cheese produced in the United States. Like propionic acid, Microgard.TM. inhibits most gram-negative bacteria and some fungi.
The Lactobacillus and Streptococcus genera also include important industrial organisms, particularly for the dairy fermentation industry. Bacteriocins produced by lactobacilli were first identified in 1961. One bacteriocin, lactacin B, is produced by Lactobacillus acidophilus N2. This bacteriocin was originally isolated as a 100,000 dalton protein complex that demonstrates bactericidal, but not bacteriolytic (bacteria dissolution) activity, against sensitive cells. The lactacin B complex dissociates to a 6,500-dalton peptide that is responsible for activity. Another bacteriocin produced by Lactobacillus helveticus 481 is helveticin J. This bacteriocin is sensitive to proteases and heat (30 min at 100.degree. C.), has a molecular weight of 300,000 daltons, and is, in its natural form, an aggregate of 37,000-dalton proteins that have bacteriocin activity.
The lactic acid-forming bacteria Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus are used to curdle milk into yogurt. These bacteria have become increasingly significant due to the rise of yogurt consumption in the United States over the past 10 years. Annual U.S. consumption is now approximately 1 billion pounds and the wholesale value for non-frozen yogurt in 1989 was $985 million. Per capita consumption in 1989 was about 4 pounds.
Generally, Americans prefer a less sour-tasting yogurt. The sour taste of yogurt arises from over-acidification caused by the above-mentioned starter bacteria strains employed to produce the yogurt. The desirable pH of yogurt is 4.3 to 4.0. During storage, yogurt bacteria, particularly Lactobacillus bulgaricus, continue to acidify yogurt and reduce the pH to well below a pH of 4.0 (sometimes as low as 3.2 to 3.5). One method by which the industry currently addresses the problem is to increase the proportion of Streptococcus thermophilus cells and decrease the proportion of Lactobacillus bulgaricus. Alternatively, the yogurt may be pasteurized to kill both the lactobacilli and streptococci. However, pasteurized yogurt has an altered flavor and lacks the living lactic and bacteria. Thus some benefits attributable to consuming yogurt that contains living cultures are lost. The need exists for a natural foodgrade organism to control the growth of the yogurt starter bacteria so that a less sour, more palatable yogurt can be obtained.